3 research outputs found
Role of G<sub>i/o</sub> proteins, Gβγ dimers and PI3K in Akt phosphorylation in response to ghrelin.
<p>A, Time-course of the effect of ghrelin on Akt HM (S473) and A-loop (T308) phosphorylation. B, Ghrelin-induced Akt phosphorylation in the absence or presence of PTX (100 ng/mL, 12 h), PI3K inhibitor wortmannin (1 µM, 30 min) and βγ sequester β-ARK-CT. In A and B, serum-starved HEK-GHSR1a cells were treated with ghrelin (100 nM) at 37°C for the indicated times. Cells were lysed and analyzed by SDS-PAGE using specific antibodies. Akt phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation obtained for each residue (mean±S.E. of three independent experiments). Blots are representative of three independent experiments.</p
c-Src is required for phosphorylation of Akt in response to ghrelin.
<p>A, Effect of siRNA depletion of c-Src on ghrelin-induced Akt phosphorylation. HEK-GHSR1a cells transfected with c-Src siRNA were serum-starved for 12 h and then stimulated with ghrelin (100 nM) at 37°C. After stimulation, equal amounts of protein in each sample were used to assess the expression of c-Src (left panel) and Akt phosphorylation (right panel) by western blotting. Expression of c-Src was quantified by densitometry and expressed as percentages of the level of c-Src in control siRNA-transfected cells (mean±S.E.). Akt phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation at HM (S473) and A-loop (T308) after ghrelin addition to control siRNA-transfected cells (mean±S.E.). B, Effect of ghrelin on Y phosphorylation of Akt and interaction between Akt and c-Src. Cells were incubated with ghrelin (100 nM, 5 min) at 37°C, lysed and immunoprecipitated (IP) with antibodies to Akt, and then analyzed by western blotting with pAkt HM(S473), pY, pSrc (Y416) antibodies. C, Effect of ghrelin on Y phosphorylation of Akt in the presence of c-Src siRNA. HEK-GHSR1a cells transfected with c-Src siRNA were serum-starved for 12 h and then stimulated with ghrelin (100 nM, 5 min) at 37°C. Equal amounts of protein in each sample were used to assess the expression of c-Src [upper panel; values shown (mean±S.E.) are percentages of the level of c-Src in control siRNA-transfected cells]. Cells were lysed and immunoprecipitated (IP) with antibodies to Akt, and then analyzed by western blotting with with pAkt HM(S473), pY, pSrc (Y416) antibodies. Immunoblots are representative of three independent experiments.</p
Octreotide-Mediated Tumor-Targeted Drug Delivery via a Cleavable Doxorubicin–Peptide Conjugate
Although
recent methods for targeted drug delivery have addressed
many of the existing problems of cancer therapy associated with undesirable
side effects, significant challenges remain that have to be met before
they find significant clinical relevance. One such area is the delicate
chemical bond that is applied to connect a cytotoxic drug with targeting
moieties like antibodies or peptides. Here we describe a novel platform
that can be utilized for the preparation of drug–carrier conjugates
in a site-specific manner, which provides excellent versatility and
enables triggered release inside cancer cells. Its key feature is
a cleavable doxorubicin–octreotide bioconjugate that targets
overexpressed somatostatin receptors on tumor cells, where the coupling
between the two components was achieved through the first cleavable
disulfide-intercalating linker. The tumor targeting ability and suppression
of adrenocorticotropic hormone secretion in AtT-20 cells by both octreotide
and the doxorubicin hybrid were determined via a specific radioimmunoassay.
Both substances reduced the hormone secretion to a similar extent,
which demonstrated that the tumor homing peptide is able to interact
with the relevant cell surface receptors after the attachment of the
drug. Effective drug release was quickly accomplished in the presence
of the physiological reducing agent glutathione. We also demonstrate
the relevance of this scaffold in biological context in cytotoxicity
assays with pituitary, pancreatic, and breast cancer cell lines